EFFICIENCY OF X-RAY TEST IN THE IDENTIFICATION OF DAMAGE AND ITS RELATION WITH
THE PUMPKIN SEEDS QUALITY
Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos,
Raquel Maria de Oliveira Pires, *Barbara Gomes Ribeiro, Edila Vilela de Resende Von Pinho,
Maria Laene Moreira de Carvalho and Carla Massimo Caldeira
Universidade Federal de Lavras, Departamento de Agricultura, Avenida Campus Universitário,
ARTICLE INFO ABSTRACT
The high quality seeds use for the pumpkin fruit production is an important
directly related to productivity. The objective of this study was to evaluate the efficiency of x in the physiological and biochemical quality of pumpkin seeds (
different lots were submitted
an intensity of 18 kV per 20 seconds. After, the seeds were separated into three different classes: full, damaged and translucent, being these submitted to germination and emergence te
replicates of 10 seeds from each lot. Tetrazolium test still was performed in the remaining seeds. For the biochemical quality assessment was verified the activity of enzymes catalase and superoxide dismutase from different types of classes an
detecting abnormalities in pumpkin seeds that negatively affect germination. The enzymes catalase expression and superoxide dismutase is directly related to the seeds quality and through these is possible to separate the lots in the same classes established by x
Copyright © 2016 Luiza do Nascimento Antônioet al.
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
INTRODUCTION
The seeds use is one of the most expensive parts in the implementation of any culture, including pumpkins, where kg is sold around R$200.00. Currently, the vegetable seed companies put on the market available to the farmer lo germination over 95%, being the seed quality important factor for the implementation and subsequent success of culture, as well as ensuring a quick and uniform cultivation and trade gains (Marcos Filho, 2015). For pumpkin fruits’ commercial production the use of high quality seeds is fundamental factor. Low productivity often is associated, among other factors, the use of seed lots with low germination, reducing vigor and high contamination by pathogens. In pumpkin crop is often the occurrence of empty seeds and damaged morpholo
latter difficult separation and identification due to the type of processing that suffer, also, by the hard tegument presence,
*Corresponding author: Barbara Gomes Ribeiro
Universidade Federal de Lavras, Departamento de Agricultura, Avenida Campus Universitário, s/n, 37200-000, Lavras
ISSN: 0975-833X
Vol.
Article History:
Received 20th November, 2015 Received in revised form 30th December, 2015
Accepted 07th January, 2016
Published online 27th February,2016
Key words:
Curcubita moschata, Image analisys, Physiological quality.
Citation: Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos, Raquel Maria de Oliveira Pires,
*Barbara Gomes Ribeiro et al, 2016. “Efficiency of x International Journal of Current Research, 8, (02), 26471
RESEARCH ARTICLE
RAY TEST IN THE IDENTIFICATION OF DAMAGE AND ITS RELATION WITH
THE PUMPKIN SEEDS QUALITY
Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos,
Raquel Maria de Oliveira Pires, *Barbara Gomes Ribeiro, Edila Vilela de Resende Von Pinho,
Maria Laene Moreira de Carvalho and Carla Massimo Caldeira
de Federal de Lavras, Departamento de Agricultura, Avenida Campus Universitário,
s/n, 37200-000, Lavras – MG
ABSTRACT
The high quality seeds use for the pumpkin fruit production is an important
directly related to productivity. The objective of this study was to evaluate the efficiency of x in the physiological and biochemical quality of pumpkin seeds (Curcubita moschata different lots were submitted to x-ray test in Faxitron x-ray device, MX20 model, DC
an intensity of 18 kV per 20 seconds. After, the seeds were separated into three different classes: full, damaged and translucent, being these submitted to germination and emergence te
replicates of 10 seeds from each lot. Tetrazolium test still was performed in the remaining seeds. For the biochemical quality assessment was verified the activity of enzymes catalase and superoxide dismutase from different types of classes and lots of seeds. The use of x
detecting abnormalities in pumpkin seeds that negatively affect germination. The enzymes catalase expression and superoxide dismutase is directly related to the seeds quality and through these is possible to separate the lots in the same classes established by x-ray test.
This is an open access article distributed under the Creative Commons Att use, distribution, and reproduction in any medium, provided the original work is properly cited.
The seeds use is one of the most expensive parts in the implementation of any culture, including pumpkins, where kg is sold around R$200.00. Currently, the vegetable seed companies put on the market available to the farmer lots with eing the seed quality important factor for the implementation and subsequent success of culture, as well as ensuring a quick and uniform cultivation and trade For pumpkin fruits’ commercial eds is fundamental factor. Low productivity often is associated, among other factors, the use of seed lots with low germination, reducing vigor and high contamination by pathogens. In pumpkin crop is often the occurrence of empty seeds and damaged morphologically, the latter difficult separation and identification due to the type of processing that suffer, also, by the hard tegument presence,
*Corresponding author: Barbara Gomes Ribeiro
Universidade Federal de Lavras, Departamento de Agricultura, 000, Lavras – MG
which impedes the embryo viewing without damaging the seeds (Carvalho et al., 2009). There are several methods that assess the seeds quality. The germination test is official and carried out in many laboratories for the physiological potential evaluation. In addition to vigor and viability tests, enzyme activity analysis, physical purity and genetic, and non destructible tests, such as image analysis of the x
(MACHADO and CICERO, 2003
advantages because it is fast, simple and non
allows the identification of internal damage seeds, viewing the embryos development phase and evaluation of seeds’ internal morphology (COPELAND and MCDONALD, 1985
the objective of this study was to evaluate the efficiency of x rays test to identify damage and its relation with the biochemical and physiological quality of pumpkin seeds.
MATERIAL AND METHODS
The research was conducted at the Seed Center Laboratory of the Agriculture Department
-(UFLA), in Lavras, MG - Brazil. Available online at http://www.journalcra.com
International Journal of Current Research
Vol. 8, Issue, 02, pp.26471-26475, February, 2016
INTERNATIONAL
Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos, Raquel Maria de Oliveira Pires, Efficiency of x-ray test in the identification of damage and its relation with the pumpkin seeds quality
26471-26475.
z
RAY TEST IN THE IDENTIFICATION OF DAMAGE AND ITS RELATION WITH
Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos,
Raquel Maria de Oliveira Pires, *Barbara Gomes Ribeiro, Edila Vilela de Resende Von Pinho,
Maria Laene Moreira de Carvalho and Carla Massimo Caldeira
de Federal de Lavras, Departamento de Agricultura, Avenida Campus Universitário,
The high quality seeds use for the pumpkin fruit production is an important factor because it is directly related to productivity. The objective of this study was to evaluate the efficiency of x-ray test
Curcubita moschata). Seeds of three
ray device, MX20 model, DC-12 option, with an intensity of 18 kV per 20 seconds. After, the seeds were separated into three different classes: full, damaged and translucent, being these submitted to germination and emergence tests using 10 replicates of 10 seeds from each lot. Tetrazolium test still was performed in the remaining seeds. For the biochemical quality assessment was verified the activity of enzymes catalase and superoxide d lots of seeds. The use of x-ray technique is effective for detecting abnormalities in pumpkin seeds that negatively affect germination. The enzymes catalase expression and superoxide dismutase is directly related to the seeds quality and through these is
ray test.
is an open access article distributed under the Creative Commons Attribution License, which permits
which impedes the embryo viewing without damaging the There are several methods that assess the seeds quality. The germination test is official and oratories for the physiological potential evaluation. In addition to vigor and viability tests, enzyme activity analysis, physical purity and genetic, and non-destructible tests, such as image analysis of the x-rays test
MACHADO and CICERO, 2003). The x-rays test has advantages because it is fast, simple and non-destructive, allows the identification of internal damage seeds, viewing the embryos development phase and evaluation of seeds’ internal
COPELAND and MCDONALD, 1985). Thus, e of this study was to evaluate the efficiency of x-rays test to identify damage and its relation with the biochemical and physiological quality of pumpkin seeds.
MATERIAL AND METHODS
The research was conducted at the Seed Center Laboratory of - Federal University of Lavras Brazil. Three lots of pumpkin seeds
INTERNATIONAL JOURNAL OF CURRENT RESEARCH
Luiza do Nascimento Antônio, Hugo Cesar Rodrigues Moreira Catão, Heloisa Oliveira dos Santos, Raquel Maria de Oliveira Pires, ray test in the identification of damage and its relation with the pumpkin seeds quality”,
Curcubita moschata being each lot characterized by seeds
produced in three different seasons were used (2003/04, 2008/09, 2013/14) produced in the Jaíba region, north of
Minas Gerais. The seeds were stored in cold storage with
average temperature 10 °C and relative air humidity around 40%, until the time of testing. Seeds of three lots were submitted to x-ray test, using the Faxitron x-ray device, MX20 model, DC-12 option, with an intensity of 18 kV per 20 seconds. The seeds were arranged on transparent blades containing 100 seeds per blade. Then, the seeds were classified and separated according to the internal structure into three distinct classes: full, translucent and damaged (Figure 1).
The seed water content was determined by oven method at 105 °C per 24 hours (BRAZIL, 2009), using 2 x 50 seeds per lot. After this period, the seeds were taken to desiccators until the cooling of the samples and then, was performed the dry mass of them. The results were expressed as a percentage. Germination test was conducted with 300 seeds of each lot, 100 seeds from each class, with 10 replicates of 10 seeds and the initial quality of each lot with 200 seeds, 8 replicates of 25 seeds. Sowing was made between paper towels type Germitest moistened with distilled water at a ratio of 2.5 ml.g-1 of paper. Soon after, they were put to germinate in germinator chamber at a constant temperature at 25 °C and evaluations of normal seedlings were made on two counts. The first on the fourth day and the last on the eighth day after sowing, according the Rules for Seed Analysis (BRAZIL, 2009). The results were expressed as average percentage of normal seedlings of the replicates. Sowing was conducted in plastic trays, 1200 cm3 containing substrate for growing vegetables. In each container were placed 5 randomly replicates. Daily evaluations were performed from the beginning of the emergency, counting the number of emerged seedlings. It was considered the percentage of normal seedlings at 21 days. To calculate the emergency speed index, according to Maguire (1962), daily readings of the number of seedlings with cotyledons leaves above the soil were carried out. The emergency speed was held along to the emergency test writing down every day and at the same time, the number of emerged seedlings until complete stabilization
of the stand. The emergency speed was determined according to the expression of Edmond and Drapala (1958).
Theremaining seeds of the germination test for each treatment were chopped longitudinally with a scalpel. These were placed
in a dark plastic container, immersed at 1% tetrazolium salt solution during 6 hours at constant temperature 30 °C. After staining, the evaluation was carried out with aid of stereoscopic microscope to determine the viability (BRAZIL, 2009). For enzyme analysis 10 seeds from each treatment were collected and macerated in the PVP presence and liquid nitrogen in melting pot onto ice and then stored at -86°C. Prior to maceration was removed the seed tegument to facilitate the enzymes extraction. For the enzymes extraction was added the extraction buffer (Tris HCL 0.2 M pH 8 + 0.1% β-mercaptoethanol) at a ratio 300 ml per 100 mg of seed powder.
The material was homogenized in vortex and kept in the refrigerator overnight followed by centrifugation at 14000 rpm per 30 minutes at 4 °C. It was applied 60 µL of supernatant onto the gel. The electrophoretic run was performed on polyacrylamide gels system 7.5% (separating gel) and 4.5% (concentrating gel). The run was performed at 120 V per 7 hours. At the end of the run, the gels were revealed for the enzymes Catalase and superoxide dismutase. The gel/electrode system used was Tris-glycine pH 8.9. Gels evaluation was performed on transilluminator and is considered the variation of bands intensity (Alfenas, 2006). The experimental design was completely randomized, factorial (3X3) analyzed three lots and three seed classes (full, damaged and translucent). Data were interpreted by variance analysis for all tests using the statistical program Sisvar® (Ferreira, 2000). To compare the averages, it was used the Scott-Knott test at 5% probability.
RESULTS AND DISCUSSION
The average water content of seeds at the time of testing was 8.3 with a maximum variation of 0.5. It is important to have a water content among lots tested with the lower variation possible, since high water content values accelerates the deterioration process and formation of products which cause immediate damage (Marcos Filho et al., 2005). In Table 1, for counting normal seedlings in the germination test it is observed that lot 1 has a lower germination of seeds damaged in relation to translucent and full seeds. Already in the lots 2 and 3 do not observe this difference. Regarding the classes noted that the full seeds, translucent and damaged of the first lot had lower germination compared to other lots of the same class. Lots 2
and 3 had a higher percentage of normal plants. In table 2 in the abnormal seedlings count it was observed that lot 1 the damaged seedlings were lower to the full and translucent classes. Lots 2 and 3 do not differ. Within the classes it is observed that in the full the lot 1 had a higher percentage of abnormal seedlings. In class damaged, showed no difference and in the translucent lot 1 had a high percentage compared to the other two lots.
Table 1. Average results of normal seedlings for three lots of pumpkin seeds and three different classes
Germination (%)
Classes Lot 1 Lot 2 Lot 3 Full 68 Ba 98 Aa 90 Aa Damaged 49 Bb 92 Aa 91 Aa Translucent 61 Ba 95 Aa 95 Aa CV(%) 7.00
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%.
Table 2. Percentage of abnormal seedlings for three lots and three pumpkin seeds classes
Abnormal seedlings (%)
Classes Lot 1 Lot 2 Lot 3 Full 25 Aa 2 Ba 7 Ba Damaged 12 Ab 5 Aa 7 Aa Translucent 31 Ba 2 Aa 2 Aa CV(%) 55.94
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%.
Table 3. Percentage of dead seeds of three lots and three pumpkin seeds classes
Dead (%)
Classes Lot 1 Lot 2 Lot 3 Full 7 Aa 0 Aa 3 Aa Damaged 39 Bb 3 Aa 2 Aa Translucent 8 Aa 3 Aa 2 Aa CV(%) 53.47
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%
In table 3, for the dead seeds, it is observed that the lot 1 the damaged seeds had a higher percentage, very high differentiating from full and translucent. Already in the lots 2 and 3 do not observe difference among the classes. Regarding the classes, the same result is observed, full and translucent did not differ in relation to the lots and in damaged treatment, lot 1 obtained a high percentage, having a worst rating. In relation to the profiles it was observed that the pumpkin seeds had reduced germination rate due to storage, and similar results have been found by Carvalho et al. (2009). Similar results indicate that damaged seeds affect performance of seed lots and were obtained in studies conducted with forest species
such as ipe tree (Oliveira et al., 2004), canephora (Oliveira
et al., 2003), mastic-white (Machado and Cicero, 2003)
and also for pumpkin seeds (Carvalho et al., 2009). In Table 4, comparing lot 1, it is observed that the full seeds and translucent had a better performance in relation to the damaged seeds. In lot 2 it is observed that the full seeds obtained better performance different of damaged and translucent. In lot 3 damaged and translucent seeds were higher than full seeds. Within the classes, full seeds of the first and second lot were higher than third. In the damaged class, it is observed that the seeds of lot 3 were higher than other two lots. And the translucent seeds, it is observed that the lot 2 was lower than lots 1 and 2.
Table 4. Emergence (%) of three lots and three pumpkin seeds classes
Emergence (%)
Classes Lot 1 Lot 2 Lot 3 Full 84 Aa 66 Aa 40 Bb Damaged 47 Bb 37 Bb 75 Aa Translucent 70 Aa 43 Bb 75 Aa CV(%) 22.16
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%.
Table 5. Emergency speed (days) for three lots and three pumpkin seeds classes
Emergency speed (days)
Classes Lot 1 Lot 2 Lot 3 Full 12 Ba 11 Ba 16 Aa Damaged 13 Ba 10 Ba 12 Bb Translucent 15 Aa 13 Ba 15 Aa CV(%) 12
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%.
In Table 5, analyzing the data within the lots 1 and 2 do not observe difference. Already on the lot damaged were statistically lower than other classes. Analyzing the full seeds, it is realized that lot 3 was statistically better than lots 1 and 2. In the damaged do not observe difference, and the translucent lot 2 was statistically lower. In table 6, analyzing the lot 1 it was observed that the full seeds were higher than other two classes. Lot 2 the full seeds were statistically higher than other two classes. Already in lot 3 the full seeds were lower than seeds of other classes. In relation to the full class, seeds lots 1 and 2 were higher than lot 3. In the damaged class, seeds lot 3 were higher than those of lot 1 and 2. And the translucent seeds do not differentiate in different lots. The emergency test on the field is important to evaluate the physiological quality of seeds for sowing and marketing purposes, which has been fundamentally based on the germination test. Lots with high homogeneity are well evaluated by the germination test, however if the heterogeneity degree is high, the vigor tests will better assess the performance of these lots at the field level (Spina and Carvalho, 1986). Delay and disuniformity of development can be reflected in product quality and reducing the commercial value as lettuce, cabbage, carrots, cauliflower, 26473 International Journal of Current Research, Vol. 08, Issue, 02, pp. 26471-26475, February, 2016
eggplant and onion (Kikuti and Marcos Filho
valid to point out that emergency results on the field can vary widely depending on edaphoclimatic conditions, even for seed lots that have high germination capacity (
2008), as observed for lot 2. It is observed in lot 1, the full seeds remaining of germination, among all only one was not feasible. Already the damaged and translucent, all were dead, represented by chart 1. In lot 2, it is seen 2 there was no remaining seeds of the full class. But the remaining seeds of the damaged and translucent classes, all were dead. In lot 3, it was observed that all remaining seeds, regardless of class, were dead. Among the available methods for assessing the seeds quality, the tetrazolium test is one of the mo
by how quickly and accuracy in determining the viability and vigor (Silva, 2006).
Table 6. Results of emergency speed index (ESI) for three lots and pumpkin seeds and three different treatments
Emergency speed index
Classes Lot 1 Lot 2 Lot 3 Full 0.68 Aa 0.77 Aa 0.25 Bb Damaged 0.36 Bb 0.32 Bb 0.73 Aa Translucent 0.46 Ab 0.34 Ab 0.52 Aa CV(%) 8.95
Average followed by the same capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by Scott-Knott test at 5%.
Figure 2. Expression pattern of catalase enzyme in terms of classes and tested seed lots, wherein (C) full seeds, (D) damaged
seeds and (T) translucent seeds
Figure 3. Expression pattern of superoxide dismutase enzyme in terms of classes and tested seed lots, wherein (C) full seeds, (D)
damaged seeds and (T) translucent seeds
By electrophoretic analysis for the enzyme catalase
seen that the first lot, the translucent seeds had a higher deterioration because it has a higher expression of this enzyme. Followed by damaged and after full seeds. Already in lot 2, it is verified that the expression of damaged is
by translucent and after full seeds. In lot 3, it is seen that three
Marcos Filho, 2007). It is still valid to point out that emergency results on the field can vary widely depending on edaphoclimatic conditions, even for seed lots that have high germination capacity (Chachalis et al.,
It is observed in lot 1, the full seeds remaining of germination, among all only one was not feasible. Already the damaged and translucent, all were dead, In lot 2, it is seen 2 there was no he remaining seeds of the damaged and translucent classes, all were dead. In lot 3, it was observed that all remaining seeds, regardless of class, Among the available methods for assessing the seeds quality, the tetrazolium test is one of the most efficient, by how quickly and accuracy in determining the viability and
(ESI) for three lots and pumpkin seeds and three different treatments
Lot 3 0.25 Bb 0.73 Aa 0.52 Aa
capital letters, on the lines, and lower in columns for each treatment and lot, do not differ by
Expression pattern of catalase enzyme in terms of classes and tested seed lots, wherein (C) full seeds, (D) damaged
and (T) translucent seeds
Expression pattern of superoxide dismutase enzyme in terms of classes and tested seed lots, wherein (C) full seeds, (D)
eeds and (T) translucent seeds
By electrophoretic analysis for the enzyme catalase may be seen that the first lot, the translucent seeds had a higher deterioration because it has a higher expression of this enzyme.
Already in lot 2, it is verified that the expression of damaged is higher, followed by translucent and after full seeds. In lot 3, it is seen that three
classes have a uniform deterioration, full, translucent and damaged seeds. Oxidative stress caused is combated by a complex antioxidant defense system, related to increa production and metalloenzymes activation, among which is the catalase (CAT), a type of very active peroxidase which removes hydrogen peroxide (H
through its conversion in water 2H
Scandalios, 2005). During the seeds deterioration process, occurs decrease of this enzyme activity, for its progressive inactivation or reduction and downtime of its synthesis (Marcos Filho, 2005). According
Sivritepe (2010), the overall decrease in C
seed decreases respiratory capacity, reducing the energy supply (ATP) and assimilated to germination thereof. When evaluated the enzyme expression SOD was possible to observe the reduction in the expression according to the seeds classes within each lot, where observed higher activity of this enzyme in full seeds, can this activity be high due to the protective role that this enzyme plays during the metabolic activation process (Figure 3). Superoxide dismutase (SOD) is a metalloenzymes group able to catalyzing the formation of hydrogen peroxide from superoxide radicals, eliminating them and releasing the cell from oxidative process. This enzyme is found in the cell cytoplasm and mitochondrial matrix (GUELFI, 2001; MCDONALD, 1999). Stewart
soybeans which viable seeds have the ability to produce SOD in the first hours of imbibition, but ones non
activity. Bailly et al. (1996) found that the loss of sunflower seed viability, which also has high oil
decrease in the superoxide dismutase activity. These results are consistent with those found by
seeds and by Sung and Jeng (1994)
Conclusion
The use of x-ray technique
abnormalities in pumpkin seeds that negatively affect germination. The expression of catalase and SOD enzymes is directly related to the seeds quality, and through these is possible to separate lots in the same classes established by x ray test.
Acknowledgements
To Personnel Improvement Coordination of Higher Education – (CAPES), to the National Council for Scientific and Technological Development (CNPq),
of the Minas Gerais State –
support and scholarships awarded to the
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